Cellulose production by facultatively anaerobic microorganisms

ABSTRACT

A method for producing bacterial cellulose, said method comprising culturing a biologically pure culture of a cellulose-producing  Proteus  strain in a liquid medium suitable for culturing facultatively anaerobic microorganisms, separating bacterial cellulose produced in said liquid medium from said liquid medium, washing said separated bacterial cellulose and drying said bacterial cellulose. The cellulose-producing  Proteus  strain is preferably a  Proteus myxofaciens  strain, preferably strain IDAC 071005-01 or strain ATCC 19692. The liquid medium is provided with a carbohydrate substrate containing at least one sugar selected from the group consisting of glucose, sucrose, fructose, lactose, xylose, and rhamnose. A bacterial cellulose product produced by culturing a biologically pure culture of a cellulose-producing  Proteus  strain in a liquid medium suitable for culturing facultatively anaerobic microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 11/289,703 filedNov. 30, 2005.

FIELD OF THE INVENTION

This invention relates to microbial production of cellulose. Moreparticularly, this invention relates to production of microbialcellulose by facultatively anaerobic microorganisms.

BACKGROUND OF THE INVENTION

It is well-known that naturally-occurring strains of the Gram-negativemicroorganism Acetobacter xylinum, also known as Gluconoacetobacterxylinum (see Yamada et al., 1998, Int. J. Syst. Bacteriol. 48:327-328),are able to produce and secrete significant quantities of cellulose whengrown under small-scale laboratory culture conditions wherein eachmicrobial cell produces a single strand of cellulose commonly referredto as a strand or a fibril. Each fibril comprises multiple inter-twistedcellulose chains or microfibrils. The biochemical basis, genetics andregulation of cellulose biosynthesis in Acetobacter xylinum have beenextensively studied, reported, and reviewed. Acetobacter spp. areobligate aerobic microorganisms, i.e., they have a strict requirementfor O₂ for respiration which drives their metabolism, growth andcellulose production. When grown in standing i.e., non-shaken/agitated,liquid cultures, Acetobacter xylinum produces pellicles comprisingdisorganized layers of long intertwined cellulose strands at theinterfaces between the air and liquid media. As the extent and thicknessof the cellulose-containing pellicle layers increase in such standingcultures, they increasingly impede then stop O₂ availability from theheadspace above the pellicles to the Acetobacter cells underlying thepellicles thus limiting and stopping cellulose production. Althoughcellulose produced by Acetobacter spp. in standing liquid cultures ischemically similar to cellulose produced from wood pulp, the majordifference is that the cross-sectional diameter of Acetobacter spp.cellulose fibrils is usually about 2 orders of magnitude smaller thancellulose fibrils from wood. Typically, the cross-sectional dimensionsof microfibrils produced by Acetobacter spp. is about 1.6 nm

5.8 nm, and they are twisted together to form fibrils (i.e., strands)having cross-sectional dimensions of about 3.2 nm×133 mm.

Cellulose production can be increased by culturing Acetobacter spp. inagitated liquid media wherein O₂ availability to individual Acetobacterspp. cells is increased through dissolved O₂ continually dispersedwithin and throughout the liquid media. Cellulose produced byAcetobacter spp. grown in such culture conditions is localized inmultiple pellets circulating throughout the media. U.S. Pat. No.4,863,565 and related U.S. Pat. Nos. 5,079,162, 5,144,162, 5,871,978,and 6,329,192 disclose that the macroscopic structure of cellulose inpellets produced by Acetobacter spp. cultured in agitated liquid mediais characterized by a three-dimensional reticulated lattice structurethat is significantly different from the layered cellulosemacrostructure produced in pellicles from standing liquid cultures. Thereticulated cellulose structure from liquid cultures is characterized byelongated strands of cellulose interconnected by shorter cellulosicbranches or filaments having cross-section diameters of 0.1μto 0.2μ,thereby forming grid-like patterns extending in three dimensions. Theformation of the shorter cellulosic branches or filaments is apparentlycaused by one or more cellulose microfibrils separating out from themain fibril produced by an Acetobacter spp. cell as a result of theconstant culture agitation. The shorter cellulosic branches interconnectand comingle with cellulose fibrils produced by other Acetobacter spp.cells thereby giving rise to the grid-like lattice structure. It alsoappears that the rates of agitation of liquid cultures significantlyaffect (a) the physical properties of the cellulose fibrils, strands,branches and filaments formed by Acetobacter spp., and (b) the degree ofinterconnecting and comingling that occurs; a low rate of agitation willresult in the formation of larger cellulose-containing pellets whileincreasingly higher rates of agitation produce increasingly smallercellulose pellets.

There are numerous problems encountered in attempting to scale celluloseproduction by Acetobacter spp. in large volumes of liquid media. Forexample, it appears that naturally occurring strains ofcellulose-producing Acetobacter spp. are unstable when cultured inshaken or agitated liquid cultures and commonly spontaneously mutateinto cellulose non-producing variants thereby limiting Acetobacter spp.cellulose production potential. As liquid culture volumes are increased,increasingly larger impellers and faster rates of impeller speeds arenecessary to produce and maintain the levels of dissolved O₂ required tosustain Acetobacter spp. respiration, metabolism and celluloseproduction. U.S. Pat. No. 4,863,565 and related U.S. Pat. Nos.5,079,162, 5,144,162, 5,871,978, and 6,329,192, and 6,329,192 teach thatshear forces in liquid media caused by high impeller speedssignificantly reduce the sizes of the three-dimensional reticulatedcellulose structures produced by Acetobacter spp. thereby substantiallydegrading the properties of the cellulose product and its commercialusefulness. Yet another problem commonly associated with celluloseproduction by Acetobacter spp. in both standing and agitated cultures isthe propensity of these microorganisms to convert glucose to gluconicacid and/or keto-gluconic acid thereby significantly dropping the pH ofthe media resulting in cessation of cellulose production. Furthermore,the conversion of glucose to gluconic and keto-gluconic acids decreasesglucose availability for cellulose production.

Strategies developed to address cellulose production problems associatedwith Acetobacter spp. include: (1) creating mutants with reducedpropensity for converting glucose into acids (e.g., U.S. Pat. No.5,079,162) or alternatively, with modified carbohydrate and/or aminoacid metabolism thereby increasing rates of cellulose production (e.g.,U.S. Pat. Nos. 5,962,278, 6,110,712 and 6,140,105), (2) addingcell-division inhibitors to modify and perhaps improve the physicalstructure and properties of cellulose produced in agitated liquidcultures (e.g., U.S. Pat. Nos. 6,060,289 and 6,627,419), (3) increasingthe availability of dissolved O₂ in large-volume vessels by combiningtwo different-shaped impellers to concurrently aerate and agitate liquidmedia (e.g., U.S. Pat. No. 6,013,490), (4) increasing O₂ availability inliquid cultures contained within vessels by increasing the amount ofaeration introduced into the vessel, thereby reducing the partialpressure of CO₂ while increasing the partial pressure of O₂ (e.g., U.S.Pat. No. 6,017,740), and (5) post-harvest processing methods forAcetobacter spp. cellulose produced in agitated liquid cultures toimprove its physical properties (e.g., U.S. Pat. No. 6,153,413).However, such strategies are complicated, costly and still have thechallenge of providing sufficient O₂ to enable optimal metabolism andcellulose production by Acetobacter spp. in large-volume liquidcultures.

It is well-known that other genera of obligate aerobic Gram-negativemicroorganisms are able to produce small amounts of cellulose fromvarious carbon substrates under carefully controlled conditions. Suchobligate aerobic cellulose-producing microorganisms include Pseudomonassp., Alcaligenes sp., Achromobacter sp., Aerobacter sp., Azotobactersp., Agrobacterium sp., and Rhizobium sp. isolated from sewage samples(Deinema et. al., 1971, Arch. Mikrobiol. 78:42-57), Rhizobium sp.isolated from leguminous plants (Napoli et al., 1975, Appl. Microbiol.30:123-131), and Agrobacterium tumefaciens (Mathysse et al., 1995, J.Bacteriol. 177:1069-1075). However, the amounts of cellulose produced bythese microbial genera are small relative to their carbon substrateinputs, and also, when compared to cellulose production by Acetobacterspp. Deinema et al. show in their FIGS. 1-6 on page 45 (1971, Arch.Mikrobiol. 78:42-57) that Pseudomonas sp., Aerobacter sp., Agrobacteriumsp., and Azotobacter sp. produced cellulose fibrils that were branched,i.e., with microfibrils extending away from the fibrils, when grown inshaken liquid cultures. They also show in FIGS. 12 and 13 on page 48,that Pseudomonas strain (V-19-Ia) grown under the same shaken liquidculture conditions, produced elongated un-branched cellulose fibrils.

More recently, cellulose production and involvement in biofilm formationhave been demonstrated in facultative anaerobic Gram negative bacteriaincluding Escherichia coli, Klebsiella pneumoniae and Salmonellaenterica (Nobles et al., 2001, Plant Physiol. 127:529-542). Thesespecies produce minute amounts of cellulose and are not expected to beof value for large-scale production.

SUMMARY OF THE INVENTION

The exemplary embodiments of the present invention, at least inpreferred forms, are directed to the production of bacterial celluloseby facultatively anaerobic microorganisms.

According to one specific embodiment of the present invention, there isprovided a biologically pure culture of a cellulose-producing strain ofProteus sp. as exemplified by Proteus myxofaciens. In a preferred form,there is provided a biologically pure culture of cellulose-producingProteus myxofaciens strain IDAC 071005-01. However, the embodiments ofthe present invention include cellulose production with Proteusmyxofaciens strain ATCC 19692.

According to another preferred embodiment of the present invention,there is provided a method for production of bacterial cellulose whereina cellulose-producing strain of Proteus sp. is cultured in a liquidmedium suitable for culturing facultatively anaerobic microorganisms,separating bacterial cellulose produced in said liquid medium from theliquid medium, washing said separated bacterial cellulose and dryingsaid bacterial cellulose.

In a preferred form, the liquid medium is provided with a carbohydratesubstrate containing therein at least one sugar selected from the groupconsisting of glucose, sucrose, fructose, lactose, xylose, and rhamnose.

In another preferred form, the liquid medium is provided with a pHselected from the range of 5 to 10, more preferably from the range of 6to 9, and most preferably in the range of 7 to 8.5.

In yet another preferred form, the liquid medium is provided with abuffer. The buffer preferably comprises at least one of a sodiumchloride buffer or a phosphate buffer, and more preferably, comprises amixture of sodium chloride and phosphate buffers.

According to one aspect of the present invention, the method forproducing bacterial cellulose comprises culturing a cellulose-producingstrain of Proteus sp. in a liquid medium suitable for culturingfacultatively anaerobic microorganisms under aerobic conditions. In apreferred form, the cellulose-producing strain is Proteus myxofaciensIDAC 071005-01. In another preferred form, the cellulose-producingstrain is Proteus myxofaciens strain ATCC 19692.

According to another aspect of the present invention, the method forproducing bacterial cellulose comprises culturing a cellulose-producingstrain of Proteus sp. in a liquid medium suitable for culturingfacultative anaerobic microorganisms under anaerobic conditions. In apreferred form, the cellulose-producing strain is Proteus myxofaciensIDAC 071005-01. In another preferred form, the cellulose-producingstrain is Proteus myxofaciens strain ATCC 19692.

According to further preferred embodiment of the present invention,there is provided a bacterial cellulose product produced by acellulose-producing strain of a Proteus sp. In a preferred form, thecellulose-producing strain is Proteus myxofaciens IDAC 071005-01. Inanother preferred form, the cellulose-producing strain is Proteusmyxofaciens strain ATCC

Deposit of the Microorganism

Samples of Proteus myxofaciens strain PARC-59 were deposited under theterms of the Budapest Treaty at the INTERNATIONAL DEPOSITORY AUTHORITYOF CANADA (IDAC) of 1015 Arlington Street, Winnipeg, Manitoba, R3E,3R2,Canada (Telephone: 204-789-6030; Facsimile: 204-789-2018). The depositwas made on Oct. 7, 2005 and was assigned accession number 071005-01.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described in conjunction with reference tothe following drawings, in which:

FIG. 1 is a graph showing the yields of purified bacterial celluloseobtained from various sugars with Proteus myxofaciens strains IDAC071005-01 and ATCC 19692;

FIG. 2 is a graph showing the effects of increasing glucose levels oncellulose production by Proteus myxofaciens strain IDAC 071005-01;

FIG. 3 is a graph showing the effects of medium pH on the production ofbacterial cellulose by Proteus myxofaciens strains IDAC 071005-01 andATCC 19692;

FIG. 4 is a graph showing the effects of a buffer on the production ofbacterial cellulose by Proteus myxofaciens strain IDAC 071005-01;

FIG. 5 is a scanning electron micrograph of the bacterial cellulosematrix produced by Proteus myxofaciens strain IDAC 071005-01 of thepresent invention;

FIG. 6 is a scanning electron micrograph of the bacterial cellulosematrix produced by Acetobacter xylinum strain PARC 017;

FIG. 7 is a one-dimensional micro-diffraction diagram showing the degreeof crystallinity of cellulose produced by Proteus myxofaciens strainIDAC 071005-01; and

FIG. 8 is a one-dimensional micro-diffraction diagram showing the degreeof crystallinity of cellulose produced by Acetobacter xylinum strainPARC 017.

DETAILED DESCRIPTION OF THE INVENTION

Facultatively anaerobic microorganisms can proliferate and produceextracellular products both in the presence and the absence of oxygen.When oxygen is present in their culture media, obligate aerobes andfacultative anaerobes will utilize the oxygen to oxidize carbohydratesthereby producing energy in the form of ATP along with substratesrequired for subsequent cellular metabolism, growth and productivity.Oxygen serves as the terminal electron acceptor in the electrontransport chain by which ATP is produced. When oxygen is limiting orabsent from their culture media, obligate aerobic microorganisms such asAcetobacter spp. are unable to oxidize carbohydrates resulting in thecessation of energy production, subsequent metabolism and growth.However, facultatively anaerobic microorganisms are able to utilize awide variety of other compounds as alternative terminal electronacceptors for the electron transport chain in a process known asanaerobic respiration. Examples of compounds that may be utilized byfacultative anaerobes for anaerobic respiration include nitrogenouscompounds such as nitrates and nitrites, sulfur compounds such assulfates, sulfites, sulfur dioxide, and elemental sulfur, carbondioxide, iron compounds, and manganese compounds. Consequently, comparedto obligate aerobic microorganisms, facultative anaerobes are easier toproduce and maintain in large-volume fermentation production systems,and do not require the same complexity of culture media and equipmentconfigurations.

According to Bergey's Manual of Systematic Bacteriology, Vol. 1 (1968),facultative anaerobic Gram-negative microorganisms generally belong toone of three families, i.e., Family 1—Enterobacteriaceae, FamilyII—Vibrionaceae, and Family III—Pasteurellaceae. The FamilyEnterobacteriaceae is made up of 13 genera which are widely distributedthroughout the world. They are commonly found in soil and water systems,and also are present as normal internal and external flora in humans andanimals. Their host range includes insects, avians, mammals, as well asplants including fruits, vegetables, grains, flowers and trees. Manyspecies of Enterobacteriaceae are pathogens of animals, avian sp.,mammals or plants and consequently, this Family has been widely studied.In general terms, Enterobacteriaceae are Gram-negative rod-shapedbacteria that are 0.3-1.0 μm×1.0-6.0 μm. Most grow well on simple mediacontaining a monosaccharide as a carbon source at temperatures in therange of 22° C. to 35° C. Escherichia coli is the type species forEnterobacteriaceae and is considered to be the most thoroughly studiedof all species of bacteria because of its ease of isolation, ease ofcultivation, rapid generation time, and its ability to be geneticallymanipulated. As a result, a wide variety of commercial fermentationprocesses, systems and culture media are known for the production ofextracellular compounds by genetically engineered E. coli for use inpharmaceutical and industrial applications.

The Genus Proteus is classified within the Enterobacteriaceae Family andcurrently consists of 5 named species, i.e., P. mirabilis, P. penneri,P. vulgaris, P. hauseri, and P. myxofaciens and three unnamedgenomospecies i.e., Proteus genomospecies 4, 5, and 6. All of the knownProteus sp. are ubiquitous in terrestrial environments, and somespecies, e.g., P. vulgaris and P. mirabilis, are common components ofthe normal intestinal flora in humans and animals. All Proteus sp. andstrains are characterized by their propensity for: (a) swarming, i.e.,movement of cells in periodic cycles of migration and consolidation,when cultured on solid agar media, and (b) production of extracellularslime.

Proteus myxofaciens is a unique but very rare specie isolated fromliving and dead gypsy moth larvae by Cosenza and Podgwaite who disclosedand characterized this species in 1966 in Antonie Leeuwenhoek J.Microbiol. Serol. Vol. 32 pages 187-191. They deposited their P.myxofaciens isolate with the American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108, U.S.A., under ascension number19692. Samples of this microorganism may be purchased directly from theATCC catalogue. Since that time, the existence and properties of the P.myxofaciens ATCC 19692 strain disclosed and made available by Cosenzaand Podgewaite have been referred to in numerous publications, but therehave been no reports of further work with and/or characterization ofthis specie nor have there been any reports of isolations of additionalstrains of P. myxofaciens.

The inventor of the present invention has surprisingly identified amicrobial strain (referred to as “PARC-59”) isolated from root and leafsurfaces of field-grown carrots, as a Proteus myxofaciens strain basedon comparisons of the morphological, biochemical and genetic profiles ofthe inventor's PARC-59 isolate with the profiles of P. myxofaciensstrain ATCC 19692. Subsequently, the inventor discovered that the P.myxofaciens PARC-59 strain produces copious amounts of cellulose whencultured in liquid media commonly used for propagation and maintenanceof microbial cultures, both under aerobic and anaerobic conditions.Accordingly, disclosed herein is a novel Proteus myxofaciens cellulosicstrain “PARC-59”. Furthermore, the inventor surprisingly discovered thatP. myxofaciens strain ATCC 19692 also produces significant amounts ofcellulose in liquid media under both aerobic and anaerobic conditions.Accordingly, disclosed herein is the use of Proteus myxofaciens strainsfor production of cellulose in liquid media under aerobic or anaerobicconditions.

Those skilled in the art will understand that the cellulosic i.e.,cellulose-producing, facultative anaerobic strains of P. myxofaciensdisclosed herein can be cultured in a wide variety of simple and complexliquid media provided with various carbon and nitrogen sources and othermineral and/or vitamin supplements useful for culturing facultativeanaerobic microorganisms. Suitable carbon sources includemonosaccharides such as glucose, fructose, and galactose, disaccharidessuch as sucrose and maltose, and complex undefined carbohydrate sourcessuch as molasses, either solely or in selected mixtures thereof.Suitable nitrogen sources include purified amino acids, nitrates,ammonium salts, and complex undefined nitrogen-rich materials such asyeast extract, malt extract, casein and corn steep liquors, eithersolely or in selected mixtures thereof.

The cellulosic strains of P. myxofaciens of the present invention may begrown in liquid media in any vessel or reactor that can be controllablyprovided with a growth media contained within the vessel or reactor.Because the cellulosic strains of the present invention are facultativeanaerobes, it isn't necessary to provide high-speed agitation togenerate turbulent flows of liquid media in order to maintain targetlevels of dissolved O₂. Accordingly, those skilled in the fermentationarts will understand how to combine and configure various types offermenter vessels and reactors with a variety of equipment includingimpellers and/or fluid circulation pumps to provide single-batch,repeated-batch, or continuous fermentation systems with suitablereplenishment of liquid media therein for cellulose production by thecellulosic facultative anaerobic microorganisms disclosed herein. Thecellulose produced in such liquid cultures may be harvested by the sameequipment and methods used in the art to harvest particulate orprecipitated extracellular products or alternatively, cellular debrisfrom other types of large-volume microbial fermentations. An example ofa preferred harvesting system is continuous centrifugation of the liquidculture through a stacked-disk multichamber centrifuge wherein a largeeffective surface area is provided for collecting thereon attachedmicroorganisms including extracellular products such as the celluloseproduced by the facultative anaerobic of the present invention. Theharvested cellulose should then be washed with water and pressed orcentrifuged to remove residual media after which the washed celluloseproduct of the present invention can be purified by heating to at least100° C. in an alkaline solution of 1N sodium hydroxide for anappropriate period of time based on the volume of cellulose productbeing purified. The alkaline solution may be removed by pressing orcontinual centrifugation and the purified cellulose washed in water,then collected and dried into its final product form.

Since both the ATCC 19692 strain of P. myxofaciens and the IDAC071005-01 strain, which were obtained from quite different sources,produce cellulose, it appears that this is a basic biological process ofP. myxofaciens that will be exhibited by all strains of themicroorganism.

The cellulosic facultative anaerobic microorganisms, methods employingsaid cellulosic facultative anaerobic microorganisms to producecellulose, and the cellulose product of the present invention aredescribed in more detail in the following examples which are intended tobe exemplary of the invention and are not intended to be limiting.

Example 1 Isolation, Identification, and Characterization of aCellulose-Producing Strain of Proteus myxofaciens

Field carrots grown at the Pacific Agri-Food Research Centre inSummerland, British Columbia, Canada, were harvested from the soil andsampled for assessments of the presence and prevalence of Listeria spp.The APHA method disclosed by Vanderzant et al. (1992, Compendium ofMethods for the Microbiological Examination of Foods. 3^(rd) Edition.American Public Health Association, Washington, D.C. pp. 637-663) wasadapted for isolation of microorganisms present on the carrots' rootsand leaves as follows. Individual root and leaf sections were subjectedto primary enrichment by incubation in test tubes containing in UVMListeria Selective broth, followed by secondary enrichment bytransferring to test tubes containing Fraser broth. Subsamples from eachtest tube containing Fraser broth were streaked onto Petri platescontaining Oxford Agar, then incubated at 25° C. for 2 days, after whichthe plates were observed for the presence of typical Listeria colonies.Presumptive Listeria isolates were purified by repeated streaking ontofresh Petri plates containing Trypticase Soy agar supplemented with 5g/L yeast extract (TSA-YE). Each purified isolate was given an ID-codenumber, and then inoculated into test tubes containing Trypticase Soybroth supplemented with 5 g/L yeast extract (TSB-YE), and incubated at25° C. for 24 h in preparation for further characterizations. Onemicrobial isolate obtained from a sample of carrot leaves and given theID-code “PARC-59”, produced a solid plug with the consistency of a gelwithin 24 h after inoculation. The plug physically displaced the liquidculture medium in the test tube after the 24 h incubation period.Another PARC-59 culture was streaked onto Petri plates containing TSA-YEagar, from which, it was inoculated into a test tube containing TSB-YEwith similar results, i.e., a solid gel plug was produced within a 24-hincubation at room temperature, and displaced the liquid medium in thetest tube.

Gram staining, oxidase and catalase tests were performed with reagentsfrom Difco (Detroit) on 24-h PARC-59 cultures grown at 25° C. on TSB-YEagar plates. The Gram reaction was verified by the KOH method. Theisolate was identified as Proteus myxofaciens with the BIOLOG automatedmicrobial identification system (Hayward, Calif.) using GN(Gram-negative) microwell plates and procedures described by themanufacturer. Proteus myxofaciens ATCC 19692, the type strain for thisspecies, was also inoculated in BIOLOG microplates and the carbonsubstrates utilized or oxidized by the two strains are shown in Table 1.Similarity indices obtained in the BIOLOG identification system were0.859 and 0.755 for the ATCC 19692 strain and the Summerland PARC-59isolate, respectively. Positive identification is considered acceptablewhen the similarity index exceeds 0.5. Accordingly, PARC-59 wasconfirmed as a P. myxofaciens strain. Proteus myxofaciens strain PARC-59is referred to herein after as Proteus myxofaciens strain IDAC 071005-01

TABLE 1 Comparisons of the substrates utlizized or oxidized by Proteusmyxofaciens ATCC 19692 and PARC-59 in BIOLOG Biolog ATCC Substratedatabase 19692 PARC-59 acetic acid − + + N-acetyl-D-glucosamine + + +cis-aconitic acid + + + alanimicide + + + L-alanine − + +L-analyl-glycine + + + L-arabinose − + − asparagine + + + asparticacid + + + bromo-succinic acid + + + 2,3-butainediol − + − citricacid + + + dextrin + + + formic acid + + + D-fructose + + +D-galacturonic acid − − + galactose + + + gentobiose + + −α-D-glucose + + + gluconic acid + + + L-glutamic acid + + +D-glucosaminic acid − − + glycerol + + + glycogen + + + D,L-α-glycerolphosphate + + + glycyl-L-aspartic acid + + + glycyl-L-glutamicacid + + + α-hydroxybutyric acid − + − myoinositol + + + α-keto butyricacid − + − α-keto glutaric acid − + − inosine + + + D,L-lacticacid + + + α-D-lactose − + + lactulose − + + L-leucine − + −maltose + + + mannose + + + glycogenmethyl pyruvate + + + mono-methylsuccinate + + + proline + + + d-psicose + + + D-serine + + +L-serine + + + D-sorbitol − + + succinamic acid + + + succinicacid + + + sucrose + + + L-threonine − + − thymidine + + +Trehalose + + + Turanose + + + Tween 40 + + + Tween 80 + + +

Example 2 Characterization of Gel Production by P. myxofaciens StrainsIDAC 071005-01 and ATCC 19692

Individual test tubes containing 10-mL of Trypticase Soy Broth (TSB),TAB amended with yeast extract (Ig/L)(TSB-YE), APT broth (APTB),Tryptose broth (TB) and Nutrient broth (NB) were inoculated with aloopful P. myxofaciens ATCC 19692 or IDAC 071005-01. One tube of eachstrain was incubated at 6° C., 20° C., 25° C., 30° C., 35° C. and 40° C.for 48 h and then examined for evidence of microbial growth and gelformation. As shown in Table 2, the relative amounts of gel produced bythe two P. myxofaciens strains during the incubation period varied withmedium composition and temperature of incubation. Gel production wasunhindered by vessel geometry and was achieved throughout deep testtubes, suggesting that synthesis of the substance was not subject tooxygen limitation. Gel formation was most extensive at 20° C. and 25° C.by both strains, but each strain produced smaller plugs comprising lesssolid, more viscous, buoyant material at higher incubation temperaturesi.e., temperatures above 25° C. Growth by both strains was observed at6° C. and at 40° C., but without secretion of gel. More gel was producedin APT broth and TSB-YE than any of the other media. Nutrient broth didnot support gel production by either strain (Table 2) suggesting thatavailability of a carbohydrate substrate was essential for gelproduction, since NB was the only growth medium that did not containglucose.

TABLE 2 Gel production by P. myxofaciens strains ATCC 19692 and IDAC071005-01 in various media at different temperatures* P. myxofaciens P.myxofaciens IDAC 071005-01 IDAC 071005-01 Growth temperature Growthtemperature Medium 20° C. 25° C. 30° C. 35° C. 20° C. 25° C. 30° C. 35°C. TSB-YE +++ +++ ++ + +++ +++ ++ + TSB ++ ++ ++ + ++ ++ + + APTB ++++++ ++ − +++ +++ ++ − TB ++ ++ ++ + ++ ++ + + NB − − − − − − − − *+++ =completely gelled ++ = thick gel plug but not completely gelled + =small gel plug − = no visible gel

The TSB-YE medium was further investigated because both strains producedgels in this medium at temperatures up to 25° C. Since the gels producedby both strains resisted boiling in 1N NaOH, a method based on thisproperty was developed for purification and quantification as shownbelow:

Clear, translucent, turgid gels swollen with water were obtained by thisprocedure. Upon freeze-drying the gel collapsed into a thin, white filmthat could be torn similar to paper. Furthermore, the dried film did notswell when immersed in water for several days at room temperature.

The effect of sugar type and quantity on the amount of purified, freezedried gel obtained by cultivation of both strains in supplemented TSB-YEis shown in Table 3.

TABLE 3 Amounts of purified gel produced by P. myxofaciens strains ATCC19692 and IDAC 071005-01 after 24-h growth at 25° C. in 100 Ml TSB-YEsupplemented with 0.1 and 1.0 g of glucose, fructose and sucrose. Eachmeasurement is the mean of four replicates Freeze-dried gel produced(mg) Substrate ATCC 19692 IDAC 071005-01 0.1 g fructose 0  3.0 ± 2.0 1.0g fructose 5.3 ± 0.9 13.1 ± 0.3 0.1 g glucose 0.3 ± 0.1  2.0 ± 1.0 1.0 gglucose 6.1 ± 0.5 19.3 ± 0.6 0.1 g sucrose 1.3 ± 0.2  7.1 ± 0.4 1.0 gsucrose 16.0 ± 1.0  29.2 ± 0.5

Gel yields increased with sugar concentrations and were highest inTSB-YE supplemented with sucrose, followed by glucose and fructose. P.myxofaciens IDAC 071005-01 produced much greater amounts of gel in eachtreatment than did ATCC 19692, regardless of sugar substrate.

Example 3 Conversion of Sugars to Cellulose by Proteus myxofaciens

The ability of P. myxofaciens to convert different types of sugars intocellulose was assessed by the following method. The following sugarswere assessed in this study: glucose, sucrose, fructose, lactose,xylose, and rhamnose. The conversion of each sugar was tested in TSB-YEliquid medium at a concentration of 10 g/L. P. myxofaciens strains IDAC071005-01 and ATCC 19692 maintained on TSA-YE agar, were inoculated intoErlenmeyer flasks containing 100-ml volumes of TSB-YE amended with theindividual sugars (3 replicates/sugar/strain). The inoculated Erlenmeyerflasks were then incubated at 25° C. for 48 h without agitation. Excessmedium was removed from the resulting mass of cells and cellulose byapplication of mechanical pressure. The mass was boiled in 1 N NaOH for2 h, and then washed three times with distilled water. The purified andwashed cellulose samples were freeze-dried prior to dry weightdeterminations. Both P. myxofaciens strains converted each sugar testedinto cellulose (FIG. 1). However, P. myxofaciens cultured in liquidmedia containing either glucose or sucrose produced the greatest amountsof cellulose (FIG. 1).

The effects of increasing sugar concentrations in liquid media oncellulose production were assessed by inoculating P. myxofaciens IDAC071005-01 into Erlenmeyer flasks containing 100-mL volumes of TSB-YEamended with one of the following concentrations of glucose: 5.0 g/L,10.0 g/L, 12.5 g/L, 15.0 g/L and 17.5 g/L. The inoculated Erlenmeyerflasks were incubated at 25° C. for 48 h. Cellulose was then harvestedfrom each flask after boiling in 1 N NaOH as described above. Thepurified and washed cellulose samples were freeze-dried prior to dryweight determinations. The data in FIG. 2 show that cellulose productionwas increased by increasing the concentration of sugar substrateprovided in the liquid medium.

Example 4 Effects of pH on Cellulose Production by Proteus myxofaciens

The effects of pH on cellulose production by P. myxofaciens wereexamined by culturing strains IDAC 071005-01 and ATCC 19692 in 100-mLvolumes TSB-YE liquid medium amended with 10 g/L glucose adjusted to pHvalues of 6.0, 7.0, 8.0, and 8.5 under conditions described above. Thedata in FIG. 3 show that both P. myxofaciens strains produced morecellulose as pH levels were increased from 6 to 8.

Example 5 Effects of Media Supplements on Cellulose Production byProteus myxofaciens

The effects of various liquid media supplements including vitamins,minerals, enzyme co-factors, and buffers such as NaCl and phosphate,were individually assessed in 100-mL volumes of TSB-YE liquid mediumamended with 10 g/L glucose, under conditions as described above.Addition of omission of various vitamins, minerals, and enzymeco-factors did not have any effects on the cellulose production by P.myxofaciens strains IDAC 071005-01 or ATCC 19692. However, the data inFIG. 4 show that optimal cellulose production was achieved when bothNaCl and phosphate buffers were incorporated into the liquid culturemedium, and that deletion of either buffer from the medium significantlyreduced cellulose production.

Example 6 Characterization and Comparisons of Cellulose Produced byProteus myxofaciens and Acetobacter xylinum

Proteus myxofaciens strain IDAC 071005-01 was cultured in 100-mL ofTSB-YE liquid medium amended with 10 g/L glucose adjusted to pH 8.5under conditions described above. Samples of gel produced in the mediumwere removed with a razor blade, then washed and sputter coated withgold and examined in a JEOL model 840 A scanning electron microscope(Japan) at an accelerating voltage of 35 kV. The micrographs revealed amatrix of highly reticulated fibres with embedded bacterial cells (FIG.5).

Acetobacter xylinum strain PARC 017 (isolated from apple cider vinegar)was cultured in static culture for 14 days at 25° C. using the mediumand method described by Hestrin and Schramm in Biochemistry Journal Vol.58 on pages 345-352 (1954). A sample of the pellicle produced on thesurface of the culture was removed and prepared for microscopicexamination as described above. A micrograph of the bacterial celluloseproduced by Acetobacter xylinum strain PARC 017 is shown in FIG. 6.Comparison of FIGS. 5 and 6 suggests that the physical structure andarchitecture of the bacterial cellulose produced by Acetobacter xylinumstrain PARC 017 is similar to that produced by Proteus myxofaciens IDAC071005-01.

The crystalline structures of the bacterial celluloses produced byProteus myxofaciens stain IDAC 071005-01 and Acetobacter xylinum strainPARC 017 were measured on a Bruker D8 Discover X-Ray diffraction unitequipped with a GADDS detector. The intensities of the samples werecollected over the range from 2θ=5 to 40. The wide-angle diffractometerwas used in transmission mode, and the measurements were performed withCuKα₁ radiation)(λ=1.54° fit with a 0.5 mm collimator and the scatteredphoton collected by a GADDS detector. The source was set to 0° while thedetector was set to 17°. The raw data were collected and fitmathematically using the method of Vonk described in Journal of AppliedCrystallography Vol. 6 on pages 148-152 (1973). The degree ofcrystallinity of the bacterial cellulose samples was expressed in termsof the crystallinity index (CrI) as defined by Segal et al. in TextileResearch Journal Vol. 29 on pages 786-794 (1959). The X-ray defractionpattern of the bacterial cellulose produced by Acetobacter xylinumstrain PARC 017 is shown in FIG. 7 while the X-ray defraction pattern ofthe bacterial cellulose produced by Proteus myxofaciens IDAC 071005-01is shown in FIG. 8. Comparisons of these data indicate that while thecrystalline structure of bacterial cellulose produced by Proteusmyxofaciens is similar to that produced by Acetobacter xylinum strainPARC 017 as evidenced by common peaks a, b, c, d, and e which weredefracted at identical Bragg Angles, it is to be noted that there may bedissimilarities between their surface architectures and topographiesbecause the CrI of the cellulose produced by Acetobacter xylinum PARC017 was 75% (FIG. 7) while the CrI of the cellulose produced by Proteusmyxofaciens was 79% (FIG. 8).

1. A biologically pure culture of Proteus myxofaciens strain IDAC071005-01.